NGS panel enhance precise diagnosis of myeloid neoplasms under WHO-HAEM5 and International Consensus Classification: An observational study

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 “triple-negative” MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.


Introduction
Myeloid neoplasms (MN) are neoplastic diseases caused by the clonal proliferation of myeloid cells, which usually involve the bone marrow and affect blood cells, thus affecting the functions of other systems and even leading to short-term death.The diagnosis of MN is complicated, mainly relying on the World Health Organization (WHO) tumor classification. [1,2]Nextgeneration sequencing (NGS), as a high-throughput technology, can sequence DNA or RNA quickly and cost-effectively, making it suitable for simultaneous detection of multiple genetic abnormalities in MN.The sequence variants/mutations it detects are widely used in MN diagnosis, prognosis, treatment decisions, and patient follow-up. [3]Since the publication of the revised 4th edition of the WHO classification of hematolymphoid tumors (WHO-HAEM4R) in 2016, NGS mutation analysis has become common in clinical practice.The basis for MN classification continues to evolve as new pathological features are discovered and new therapies become available.Therefore, the World Health Organization released the fifth edition of the World Health Organization Classification of Hemolymph Neoplasms (WHO-HAEM5) on June 22, 2022. [4]Shortly thereafter, the Clinical Advisory Committee launched the International Consensus Classification (ICC) of hematologic neoplasms. [5]he ICC classification of specific MN categories was then introduced in detail, such as acute myeloid leukemia (AML), [6] myelodysplastic syndrome (MDS), [7] myeloproliferative neoplasm (MPN), [8] myelodysplasia/myeloproliferative neoplasms (MDS)/MPN), [9] published in Virchows Arch.Therefore, WHO-HAEM4R was updated to 2 different versions at the same time.There are inevitably differences between the 2, and both the classification algorithm and the classification results need to be fully understood. [10]To this end, we used WHO-HAEM5 and ICC to reclassify MN cases previously diagnosed according to WHO-HAEM4R to explore the practical significance of these 2 latest classification systems.We endeavored to carefully examine the contribution of these NGS-detected mutation signatures to both classifications.This is very useful for everyone to understand the different effects of the 2 new classifications in practical applications.

Study population
This study included 112 patients with MN diagnosed at the Hematology Department of the Fourth Affiliated Hospital of Zhejiang University School of Medicine from May 2018 to September 2023.The cohort comprised 54 males and 58 females, with a median age of 61 years (range, 18-85 years) (Table 1).All patients underwent a comprehensive diagnostic workup, including complete blood count, white blood cell differential, bone marrow cell morphology, conventional cytogenetics, and mutation analysis using a hematological malignancy NGS panel.Additionally, some patients underwent ancillary studies such as flow cytometry immunophenotyping, fluorescence in situ hybridization, polymerase chain reaction-based fusion gene or mutation analysis, and bone marrow biopsy.The exclusion criteria included patients with incomplete information on bone marrow cell morphology, NGS panel analysis, genetic analysis, and patients who did not meet the WHO-HAEM4R MN diagnostic criteria. [11]The clinical characteristics of patients are presented in Table 1.

Sequencing and variant annotation
Genomic DNA was extracted from bone marrow or blood samples using the Maxwell RSC Blood DNA Kit (Promega et al., USA).Libraries were prepared using an enrichment-capture gene panel following the manufacturer protocol using 50 ng of DNA.A total of 141 genes associated with hematological diseases were examined, with a mean sequencing depth of 800×.Single nucleotide variants and short fragment indels in proteincoding sequences or exonic hotspots were analyzed using the Ion Reporter™ and Variant Reporter pipelines.Annotation was performed by referencing the dbSNP, 1000 Genomes, Polyphen-2, and COSMIC databases.
These 3 classifications are roughly equivalent in classifying these cases, with some differences in terminology.A total of 11 cases had significantly different classifications, including 1 case with only 12% blasts, classified by WHO-HAEM4R as MDS-EB2.In contrast, WHO-HAEM5 and ICC classified it as AML-DGA and AML-RGA, respectively, due to NPM1 mutations.Two cases were classified as AML-MRC (WHO-HAEM4R) or AML-MR (WHO-HAEM5) due to complex karyotypes; however, in ICC, they were classified as AML-TP53 because of TP53 mutation.Two cases of AML-NOS (WHO-HAEM4R) were classified as AML-MR (WHO-HAEM5) and AML-MRC (ICC) because of ASXL1 and SRSF2 mutations and ASXL1 and U2AF1 mutations, respectively.One case of MDS-MLD was classified as MDS-SF3B1 in the 2 new classifications because of SF3B1.One MDS/MPN-RS-T case and 1 MDS/ MPN-U case were reclassified as MDS/MPN-SF3B1-T because of SF3B1 mutation detected (Table 2, Figure 1).
Figure 2 provides a comprehensive view of the gene mutation landscapes within each patient with MN, considering the WHO-HAEM5 reclassified disease categories. [4,12]Genes were further organized into distinct functional groups, adapted from the relevant literature. [13]When interpreting gene mutation status, it is worth noting that in addition to pathogenic mutations, some mutations of uncertain significance, often with multiple coexisting mutations, are often found in MPN (Table 3, Figure 2).

Discussion
WHO-HAEM5 and ICC are both updates to WHO-HAEM4R.Therefore, 2 new systems are now available for classifying hematolymphoid tumors.This brings inevitable confusion to the clinical diagnosis and subclassification. [14]Comparing the 2 classifications in practice can remind us of situations that require attention.After our reclassification, we found limited differences between the 2 new classifications.The main difference was the addition of disease subtypes, defined by genetic mutations.According to the existing literature, in addition to the differences in this study, there are also some other differences between the 2, such as: The cutoff for blast cells in some AML subtypes differs between the 2 systems.However, WHO-HAEM5 eliminates the 20% blast requirement for AML by defining genetic abnormalities (except AML with BCR::ABL1 fusion, AML with CEBPA mutation, AML-MR, and AML with other defined genetic alterations). [4]n ICC, blasts must reach 10% or more, except in AML with BCR::ABL1 fusion, AML-TP53, AML-MRGM, AML-MRCA, and AML, NOS. [5]Some AML subtypes require ≥ 10% blast cells in ICC but ≥ 20% in WHO-HAEM5, such as AML with CEBPA mutations, AML with RUNX1T3(CBFA2T3)::GLIS2, AML with KAT6A::CREBBP, AML with FUS::ERG, AML with MNX1::ETV6, and AML with NPM1::MLF1.In ICC, there are genetic abnormalities such as t(1;3)(p36.3;q21.3)/PRDM16::RPN1, t(10;11)(p12.3;q14.2)/PICALM::MLLT10,t(16;21)(q24.3;q22.1)/RUNX1::CBFA2T3,and ≥ 10% blast cells can diagnose AML subtypes, which are not recognized by the WHO. [14]ML-TP53 is a unique subtype of ICC that is not recognized as an entity by the WHO-HAEM5.In ICC, as a type of AML with specific genetic abnormalities, its diagnostic priority is between AML-NPM1 and AML-MRGM.If a patient had TP53 and NPM1 mutations simultaneously without other recurrent genetic abnormalities, the patient was diagnosed with AML with NPM1 and TP53 mutations.If TP53 mutation is present along with myelodysplasia-related gene mutations, AML-TP53 should be made. [5]urthermore, in ICC, pure erythroid leukemia is often associated with TP53 mutations, and these cases are now classified as AML-TP53.However, this classification appears to lack uniqueness, as the clinical features of TP53-mutated AML are much broader than the pure erythroid leukemia, which are more homogeneous. [15] our study, there was no substantial variance in the classification of MPN across the 3 classification systems.Among most triple-negative MPN patients, NGS studies typically identified 2 or more gene mutations concurrently, although occasionally only 1 mutation was discerned (4/17).The mutation profile of  triple-negative MPN differs markedly that of MDS and AML.These mutated genes are not typically found in AML or MDS, and their clinical implications are commonly denoted as having "uncertain significance."Nevertheless, as a clonal marker, it can serve as a surrogate indicator for JAK2, CALR, or MPL mutations, which is one of the major criteria for ET and PMF. [16]he primary impact of NGS mutation screening on the diagnosis of MDS/MPN lies in the identification of SF3B1 mutations, which define genetic abnormalities.
Given the retrospective nature of this study, NGS was selectively performed in patients deemed clinically pertinent, resulting in a relatively modest sample size.The patient distribution exhibited certain biases, such as a higher proportion of triplenegative MPN cases and the absence of myeloid/lymphoid neoplasms with eosinophilia and defining gene rearrangement.However, owing to the inclusion of suspected patients for whom NGS holds the utmost relevance in classification, it achieves maximum efficiency.This underscores the impact of NGS on MN classification to a significant extent.

Conclusions
Gene mutation analysis of bone marrow samples by the hematological malignancy NGS panel is instrumental for understanding the gene mutation in MN patients and for diagnosing and classifying diseases.Therefore, MN patients is recommended to be tested as much as possible.

Table 1
Clinical characteristics of the 112 patients with myeloid neoplasms (WHO-HAEM4R).

Table 2
Defining genetic abnormalities mutations detected by NGS in AML, MDS, and MDS/MPM.
ET = essential thrombocythemia, PMF = primary myelofibrosis, PV = polycythemia vera.a The mutation is not detected by MPN gene panel.